Amanitin derivatives are prepared and characterized for their ability to inhibit the class I, II, and III eukaryotic RNA polymerases. Derivatives that show polymerase inhibition at low concentrations (Ki less than 10 to the minus 6th power M) are examined by ultraviolet and proton magnetic resonance spectroscopy to verify their structure. High specific activity radioisotopic derivatives are subsequently prepared for use in a ligand binding assay for RNA polymerase. Other types of amanitin derivatives are selected on the basis of RNA polymerase inhibition plus suitability for application to affinity chromatography techniques. Particular emphasis is placed on identifying derivatives exhibiting enhanced inhibitory activity on RNA polymerase III or detectable activity on RNA polymerase I. These various amanitin derivatives serve as a tool to quantitate the frequency and affinity of RNA polymerase binding sites on the DNA template. RNA polymerase II is quantitated by ligand binding with iodo-amanitin; this binding is specific and stable under various experimental conditions. The effect of DNA integrity, chromosomal proteins, metal ions, and polyamines on RNA polymerase II binding to DNA is studied by means of the iodo-amanitin ligand binding assay.